CN105588936A - Determination reagent for glycocholic acid and preparation method of determination reagent - Google Patents

Abstract

The invention relates to a determination reagent for glycocholic acid and a preparation method of the determination reagent, and belongs to the technical field of biochemical means testing. The determination reagent comprises an R1 reagent, an R2 reagent and a glycocholic acid standard sample; a solution formed by dissolving a sulfo-oxidation type coenzyme into purified water added with a buffer solution is adopted as the R1 reagent; a solution formed by dissolving a reduction type coenzyme into purified water added with a buffer solution, glycocholic acid dehydrogenase and sodium azide is adopted as the R2 reagent; a solution formed by dissolving a sodium glycocholate hydrate, a buffer solution and sodium azide into purified water is adopted as the glycocholic acid standard sample. The determination reagent is applied to glycocholic acid determination and has the advantages of being quick, sensitive, good in accuracy, high in specificity, good in stability and the like.

Description

Mensuration reagent of a kind of glycocholic acid and preparation method thereof

Technical field

The present invention relates to mensuration reagent of a kind of glycocholic acid and preparation method thereof, belong to biochemical means technical field of measurement and test.

Background technology

Serum CG (Cholyglycine, CG) is one of mating type cholic acid of being combined into of cholic acid and glycine,In liver cell, cholesterol, through extremely complicated enzymatic reaction, is transformed into elementary bile acid. Wherein there are cholic acid (CA) and goose deoxidationCholic acid (CD-CA). On the steroids core of cholic acid, have three hydroxyls (C3, C7, C12), the hydroxyl of side chain terminal is with peptide bond and sweet ammoniaAcid combination, molecular weight is 462u.

CG is synthetic liver cell, enters gall-bladder storage through bile duct, and gallbladder contraction after the meal, enters small intestine in company with bile, participates inDigesting and assimilating of fat, 95% cholic acid, by the heavy absorbed into serum of mucous membrane of small intestine, is delivered to liver through portal vein and is absorbed by liver cell.Under normal circumstances, liver can effectively absorb more than 99% CG in portal vein and again secrete into bile, only has 1%CG to overflow into body and followsRing. In the time that liver cell is impaired, liver cell can not effectively absorb the CG that reaches liver through the circulation of intestines liver, causes CG increased content in blood;When cholestatis, liver is drained cholic acid generation obstacle and is backflowed in blood circulation, also can make CG increased content in blood.

Clinical proof: in the biochemical indicator of hepatic injury, CG is more responsive than the test such as glutamic-pyruvic transaminase, bilirubin, concrete asUnder:

(1) in illnesss such as oxyhepatitis, CAH, primary carcinoma of liver, cirrhosis and chronic persistant hepatitisesIn, the level of its change of serum C G is all higher than Normal group, and rising positive rate successively decreases by said sequence, especially chronic in clinical discriminatingActive hepatitis and chronic persistant hepatitis aspect, the amplitude that chronic active hepatitis CG raises and frequency are all higher than chronic persistent hepatitis, and CG can be used asA valuable index, to judging progress and the alleviation of CAH, prediction curative effect and recurrence all have fine value.

(2) the pathology development process of Serum CG content rising degree and cirrhosis is proportionate, and is clinical judgment liverThe good index of hardenability and prognosis.

(3) Serum CG level significantly raises at Patients with Cholelithiasis, can be up to 50 times.

(4) when obstructive liver disease, CG level increases 10-20 doubly, and Drug Cholestasis Bile Acid in Serum also increases.

(5) pregnant woman, in the conceived middle and later periods, due to baby's increase, can oppress liver, and liver excretion cholic acid is hinderedHinder and cause the higher of CG, severe patient may cause stillborn foetus.

(6) intrahepatic cholestasis of pregnancy (Intrahepaticcholestasisofpregnancy, ICP) againClaim pregnant Delayed onset cholestasis, belong to one of high risk pregnancy. Although this disease, without pregnant woman's death, has a strong impact on fetus, easilyPremature labor occurs, and fetal distress in uterus even encloses died.

The method of current domestic mensuration glycocholic acid mainly contains:

(1) Immunoturbidimetry, the method sensitivity is lower, is prone to prozone phenomenon.

(2) chemoluminescence method, although the method is highly sensitive, unstable, repeatability is poor.

(3) ELISA, the quantitative poor accuracy of the method, the operating time is long, and automaticity is low, general only for fixedProperty detects.

Based on this, make the application.

Summary of the invention

The above-mentioned defect existing in test process in order to overcome existing glycocholic acid, first the present invention provides a kind of spirit fastQuick, accuracy good, specificity is high, good stability, easy and simple to handle, is applicable to clinical full-automatic or sweet courage that semiautomatic biochemistry is analyzedAcidity test reagent.

For achieving the above object, the technical scheme that the present invention takes is as follows:

A kind of glycocholic acid is measured reagent, comprises R1 reagent, R2 reagent and glycocholic acid standard sample, and described R1 reagent is by sulphurBe dissolved in the solution forming in the purified water that is added with buffer solution for NAD; Described R2 reagent is by reduced formDPN is dissolved in the solution forming in the purified water that is added with buffer solution, glycocholic acid dehydrogenase, Sodium azide; Described sweet courageAcid standard sample is to be dissolved in by sodium glycocholate hydrate, buffer solution and Sodium azide the solution forming in purified water.

Further, as preferably:

Described buffer solution is phosphate buffer or trishydroxymethylaminomethane, preferred, and described phosphate is slowThe pH that rushes liquid is 7.0, and concentration is 0.65mol/L.

Described Thionicotinamide-NAD I is β-Thionicotinamide urea purine dinucleotides oxidized form (Thio-NAD), itsConcentration is 1g/L.

Described DPNH is β-niacinamide urea purine dinucleotides reduced form (NADH), and its concentration is 6g/L.

Described glycocholic acid dehydrogenase concentration >=5000U/L.

Described Sodium azide concentration is 0.6mol/L.

Meanwhile, the present invention also provides a kind of preparation method with above-mentioned feature glycocholic acid mensuration reagent, comprises as followsStep:

(1) R1 reagent preparation: add buffer solution in purified water, be stirred to completely and dissolve; Add Thionicotinamide-NAD IBe stirred to completely and dissolve, constant volume after continuing to stir;

(2) R2 reagent preparation: add buffer solution in purified water, be stirred to completely and dissolve; Add glycocholic acid dehydrogenase, stirMix to dissolving; Add Sodium azide and be stirred to dissolving, add DPNH to be stirred to completely and dissolve, constant volume after continuing to stir;

(3) glycocholic acid standard sample preparation: add sodium glycocholate hydrate in purified water, be stirred to completely and dissolve; AddBuffer solution is stirred to dissolving, adds Sodium azide to be stirred to completely and dissolves, constant volume after continuing to stir;

(4) the R1 reagent to above-mentioned preparation, R2 reagent and glycocholic acid standard sample carry out sensitivity, the range of linearity, precisionMeasure with the degree of accuracy.

Further, as preferably:

Described mixing speed is 450 revs/min.

In step (1), described purified water is 1500ml, and constant volume is 1600ml.

In step (2), described purified water is 300ml, and constant volume is 400ml.

In step (3), described purified water is 80ml, and constant volume is 100ml.

Operation principle of the present invention is as follows:

Glycocholic acid in glycocholic acid standard sample is by glycocholic acid dehydrogenase (GCDH) and β-Thionicotinamide urea purine dinucleotideAcid oxidase type (Thio-NAD) is oxidized specifically, generates steroids and the reduction of β-Thionicotinamide urea purine dinucleotidesThio-NADH; And the steroids generating is under GCDH and β-niacinamide urea purine dinucleotides reduced form (NADH) effect, raw againBecome glycocholic acid and β-niacinamide urea purine dinucleotides oxidized form (NAD). By above-mentioned circulation, the bile acid of trace in serumMeasured amplification, also amplification on year-on-year basis of Thio-NADH, so by measuring Thio-NADH in the specific absorbance of 405nmChange, can calculate the content of glycocholic acid in sample.

In above-mentioned operation principle, its course of reaction is specific as follows:

(1) sensitivity evaluation test

According to wanting of in GB/T26124-2011 " clinical chemistry external diagnosis reagent (box) ", " sensitivity for analysis " being detectedAsk, under regulation parameter, sensitivity for analysis evaluation is detected to the absorbance producing with sample with kit and change, be scaled nThe difference (Δ A) of the absorbance of unit is as sensitivity for analysis. Sensitivity for analysis evaluation test is to sensitivity for analysis evaluation sampleThis replication 20 times. Assessment result: sensitivity for analysis 2.7mg/L absorbance rate of change (Δ A/min) >=0.00405ABS/min。

(2) range of linearity evaluation test

According to GB/T26124-2011 " clinical chemistry external diagnosis reagent (box) " with " external diagnosis reagent analytical performance is commentedEstimate guideline---the range of linearity (exposure draft) " in suggestion, when the reagent range of linearity is verified, to be verifiedIn the range of linearity, select 5 concentration levels, each concentration level replication 3 times.

(3) precision evaluation test

Precision evaluation comprises repeatability and difference between batch, and at least assesses the precision of two concentration level samples, whereinThere is a concentration in medical science decision level left and right. Therefore, reproducibility adopts under repeated condition, with two concentration levelsControl material (one of them concentration approaches medical science decision level) test, each concentration retest 10 times; Difference between batch is evaluatedThe control material (one of them concentration approaches medical science decision level) of two concentration levels of employing is tested respectively 3 different lot numbersKit, each lot number test 3 times.

(4) accuracy estimating test

With the people source sample that is no less than 40 variable concentrations within the scope of detectable concentration, using the analytical system of specifying asComparison method, every duplicate samples detects respectively by test agent method of operating and comparison method. Calculate two groups with linear regression methodThe coefficient correlation (r) of result and the relative deviation of each concentration point.

Assessment result: repeated coefficient of variation CV≤6% of three batches of reagent; Difference between batch≤10%; Relative deviation≤15%,Reagent linearity can reach 80mg/L, sensitivity for analysis 2.7mg/L absorbance rate of change (Δ A/min) >=0.00405ABS/min.

The present invention utilizes enzyme round-robin method to measure the method for glycocholic acid, and its advantage is rapid sensitive, and accuracy is good, specificityHeight, good stability, easy and simple to handle, support the use applicable to clinical full-automatic or semi-automatic biochemical analyzer.

Detailed description of the invention

The present embodiment main agents used: phosphate buffer: pH7.0,0.65mmol/L; Thio-NAD (sulfo-oxidationType DPN): 1g/L; Glycocholic acid dehydrogenase (GCDH): >=5000U/L: Sodium azide: 0.6mmol/L; NADH (reduced coenzymeI): 6g/L; All purchased from Enzymaker company.

1) preparation of R1 reagent

1. 1500ml purified water is joined in appropriate containers.

2. open electric mixer, mixing speed is 450 revs/min.

3. take Tris1g and add in above-mentioned purified water, be stirred to completely and dissolve.

4. take 1g Thionicotinamide-NAD I and add in above-mentioned solution, be stirred to completely and dissolve.

5. stir more than ten minutes.

6. be settled to 1600ml.

2) preparation of R2 reagent

1. 300ml purified water is joined in appropriate containers.

2. open electric mixer, mixing speed is 450 revs/min.

3. take 0.3gTris and add in above-mentioned purified water, be stirred to completely and dissolve.

4. take 2g glycocholic acid dehydrogenase and add in above-mentioned solution, be stirred to completely and dissolve.

5. take 1.2g Sodium azide and add in above-mentioned solution, be stirred to completely and dissolve.

6. take 12g DPNH and add in above-mentioned solution, be stirred to completely and dissolve.

7. stir more than ten minutes.

8. be settled to 400ml.

3) preparation of standard items

1. 80ml purified water is joined in appropriate containers.

2. open electric mixer, mixing speed is 450 revs/min.

3. take 0.8mg sodium glycocholate hydrate and add in above-mentioned purified water, be stirred to completely and dissolve.

4. take 5mgTris and add in above-mentioned purified water, be stirred to completely and dissolve.

5. take 6.7mg Sodium azide and add in above-mentioned solution, be stirred to completely and dissolve.

6. stir more than ten minutes.

7. be settled to 100ml.

4) reagent test

1. instrument configuration: instrument uses Hitachi's 7100 automatic clinical chemistry analyzers.

Location parameter is set in instrument in strict accordance with product description, and basic location parameter is as follows:

Method: end-point method, the Direction of Reaction: upwards; Wavelength: 600nm, 37 DEG C of temperature.

Cuvette optical path: 1cm; R1 reagent: 200 μ l, R2 reagent: 50 μ l, glycocholic acid standard sample: 6 μ l.

The first photometry point: read for 1 minute after R2 adds; The second photometry point: read for 5 minutes after R2 adds.

2. reagent visual examination: visual inspection.

3. loading quantity inspection: use general gage measuring.

4. reagent blank is measured:

As blank sample test kit, under test dominant wavelength, record test starting by distilled water or deionized waterTime absorbance (A1 reagent) and the absorbance after approximately 5 minutes (A2 reagent), A2 is the blank absorbency of work reagent.

5. the range of linearity is measured:

Get the sample that the high value sample physiological saline doubling dilution that approaches the range of linearity upper limit is mixed with variable concentrations gradientThis series. Calculate desired value with H value and dilution ratio. Dilution process with reference to shown in table 1 (according to the corresponding tune of large I of high valueWhole dilution gradient).

Table 1 sample dilution table

Embodiment	1	2	3	4	5
						Physiological saline (ml)	0	0.5	0.5	0.5	0.5
High value sample H (ml)	1	0.5	0.5	0.5	0.5
						Dilution ratio	Former times	1/2	1/4	1/8	1/16

In table: the concentration of embodiment 1 sample is known definite value CH, embodiment 2-5 concentration of specimens is pressed formula: concentration of specimens=CH × dilution ratio, calculates as desired value, and the series of samples of having diluted is fully mixed, and in the mensuration system after correction, pressesMeasure 2 times from low value to high value sequential parallel, as corresponding embodiment sample measured value, (measurement result has obviously average as 2 timesDeviation should be rejected and resurvey).

Statistical analysis: taking desired value as abscissa, sample measured value is ordinate mapping and linear regression analysis, determinesBetween linear zone, calculate linear equation y=a+bx and coefficient correlation, be linear good in the time of correlation coefficient r >=0.990.

Data analysis is processed and is adopted MicrosoftExcel software.

Correlation formula is as follows:

$b=\frac{{\mathrm{n\ΣX}}_{i}Y-{\mathrm{\ΣX}}_i\·{\mathrm{\ΣY}}_i}{{{\mathrm{n\ΣX}}_i}^2-{\left({\mathrm{\ΣX}}_i\right)}^2}---\left(1\right)$

$\left|a\right|=\frac{|{\mathrm{\ΣY}}_i-{\mathrm{b\ΣX}}_i|}{n}---\left(2\right)$

$r=\frac{{\mathrm{n\ΣX}}_i{Y}_i-{\mathrm{\ΣX}}_i\·{\mathrm{\ΣY}}_i}{\sqrt{\[n\·\mathrm{\Σ}Xi-\left(\mathrm{\Σ}Xi\right)\]\[n\mathrm{\Σ}Yi-\left(\mathrm{\Σ}Yi\right)\]}}---\left(3\right)$

In formula, C: concentration; V: volume; B: the slope of the tropic; ∣ a ∣: the absolute value of tropic intercept; R: coefficient correlation;X_i: each pipe desired value; Y_i: each pipe measured value; I:1,2,3. ..., n; N: measure sample number.

6. precision is measured:

A. repeatability is measured:

By clinical samples or quality controlled serum test kit, retest 10 times, the mean value of computation and measurement value

And standard deviation (s). Be calculated as follows the coefficient of variation (CV).

With coefficient of variation CV (%)

$\stackrel{\‾}{X}={\mathrm{\ΣX}}_1/n---\left(4\right)$

CV＝ $\sqrt{\frac{\mathrm{\Σ}(\stackrel{\‾}{X}-Xi)}{{\left(\stackrel{\‾}{X}\right)}^2(n-1)}}---\left(5\right)$

In formula:---the average of test serum sample;

X_i---measure the measurement result of serum sample;

N---measure number of times

CV---the coefficient of variation

B. difference between batch is measured

Get three lot number censorship reagent, each lot number is got 3 bottles, measures respectively 1 part of clinical sample or quality controlled serum, can selectRoche quality-control product, calculates respectively 9 parts of reagent and measures averageMensuration average with 3 parts of reagent of each lot numberAnd the coefficient of variation of measuring with three lot number reagent of MicrosoftExcel software statistics.

Correlation formula is as follows:

In formula:

In maximum;

In minimum of a value;

---grand mean.

7. accuracy is measured:

Get calibration object (having card reference material, CRM) kit is tested, duplicate detection 3 times, gets test result average(M), calculate relative deviation (B) by formula (7).

Relative deviation (B)=(M-T)/T × 100% ... (7)

In formula: T---there is card reference material (CRM) sign value; M---sample determination result average;

8. sensitivity for analysis:

With concentration known or active sample test kit, be recorded in kit and specify that the absorbance producing under parameter changesBecome, be scaled the absorbance difference (Δ A) of n unit.

Above content is provided technical scheme is done in conjunction with the preferred embodiment of the invention further detailedDescribe in detail brightly, can not assert that the invention is concrete to implement to be confined to above-mentioned these explanations, for technology under the inventionThe those of ordinary skill in field, without departing from the concept of the premise of the invention, can also make some simple deductionsOr replace, all should be considered as belonging to the protection domain of the invention.

Claims (10)

1. glycocholic acid is measured a reagent, it is characterized in that: comprise R1 reagent, R2 reagent and glycocholic acid standard sample, described R1Reagent is to be dissolved in by Thionicotinamide-NAD I the solution forming in the purified water that is added with buffer solution; Described R2 reagentTo be dissolved in by DPNH the solution forming in the purified water that is added with buffer solution, glycocholic acid dehydrogenase, Sodium azide;Described glycocholic acid standard sample is to be dissolved in by sodium glycocholate hydrate, buffer solution and Sodium azide the solution forming in purified water.

2. a kind of glycocholic acid as claimed in claim 1 is measured reagent, it is characterized in that: described buffer solution is phosphate-bufferedLiquid or trishydroxymethylaminomethane.

3. a kind of glycocholic acid as claimed in claim 2 is measured reagent, it is characterized in that: the pH of described phosphate buffer is7.0, concentration is 0.65mol/L.

4. a kind of glycocholic acid as claimed in claim 1 is measured reagent, it is characterized in that: described Thionicotinamide-NAD I isβ-Thionicotinamide urea purine dinucleotides oxidized form, its concentration is 1g/L.

5. a kind of glycocholic acid as claimed in claim 1 is measured reagent, it is characterized in that: described DPNH is β-cigaretteAcid amides urea purine dinucleotides reduced form, its concentration is 6g/L.

6. a kind of glycocholic acid as claimed in claim 1 is measured reagent, it is characterized in that: described glycocholic acid dehydrogenase concentration >=5000U/L。

7. a kind of glycocholic acid as claimed in claim 1 is measured reagent, it is characterized in that: described Sodium azide concentration is0.6mol/L。

8. a preparation method for glycocholic acid mensuration reagent described in claim 1-7 any one, is characterized in that, comprises following stepRapid:

(1) R1 reagent preparation: add buffer solution in purified water, be stirred to completely and dissolve; Add Thionicotinamide-NAD I to stirTo dissolving completely, constant volume after continuing to stir;

(2) R2 reagent preparation: add buffer solution in purified water, be stirred to completely and dissolve; Add glycocholic acid dehydrogenase, be stirred toDissolve; Add Sodium azide and be stirred to dissolving, add DPNH to be stirred to completely and dissolve, constant volume after continuing to stir;

(3) glycocholic acid standard sample preparation: add sodium glycocholate hydrate in purified water, be stirred to completely and dissolve; Add bufferingLiquid is stirred to dissolving, adds Sodium azide to be stirred to completely and dissolves, constant volume after continuing to stir;

(4) the R1 reagent to above-mentioned preparation, R2 reagent and glycocholic acid standard sample carry out sensitivity, the range of linearity, precision and standardExactness is measured.

9. glycocholic acid as claimed in claim 8 is measured the preparation method of reagent, it is characterized in that: described mixing speed is450 revs/min.

10. glycocholic acid as claimed in claim 8 is measured the preparation method of reagent, it is characterized in that: in step (1), describedPurified water is 1500ml, and constant volume is 1600ml; In step (2), described purified water is 300ml, and constant volume is400ml; In step (3), described purified water is 80ml, and constant volume is 100ml.